Acceptor Specificity of Different Length Constructs of Human Recombinant α1,3/4-Fucosyltransferases
1995; Elsevier BV; Volume: 270; Issue: 15 Linguagem: Inglês
10.1074/jbc.270.15.8712
ISSN1083-351X
AutoresTheodora de Vries, Cheryl A. Srnka, Monica M. Palcic, Stuart J. Swiedler, Dirk H. van den Eijnden, Bruce A. Macher,
Tópico(s)Galectins and Cancer Biology
ResumoThe acceptor specificity of recombinant full-length, membrane-bound fucosyltransferases, expressed in COS-7 cells, and soluble, protein-A chimeric forms of α1,3-fucosyltransferase (Fuc-T) III, Fuc-TIV, and Fuc-TV was analyzed toward a broad panel of oligosaccharide, glycolipid, and glycoprotein substrates. Our results on the full-length enzymes confirm and extend previous studies. However, chimeric Fuc-Ts showed increased activity toward glycoproteins, whereas chimeric Fuc-TIII and Fuc-TV had a decreased activity with glycosphingolipids, compared to the full-length enzymes. Unexpectedly, chimeric Fuc-TV exhibited a GDP-fucose hydrolyzing activity.In substrates with multiple acceptor sites, the preferred site of fucosylation was identified. Fuc-TIII and Fuc-TV catalyzed fucose transfer exclusively to OH-3 of glucose in lacto-N-neotetraose and lacto- N-tetraose, respectively, as was demonstrated by 1H NMR spectroscopy. Thin layer chromatography immunostaining revealed that FucT-IV preferred the distal GlcNAc residue in nLc6Cer, whereas Fuc-TV preferred the proximal GlcNAc residue. Incubation of Fuc-TIV or Fuc-TV with VI3NeuAcnLc6Cer resulted in products with the sialyl-LewisXepitope as well as the VIM-2 structure.To identify polar groups on acceptors that function in enzyme binding, deoxygenated substrate analogs were tested as acceptors. All three Fuc-Ts had an absolute requirement for a hydroxyl at C-6 of galactose in addition to the accepting hydroxyl at C-3 or C-4 of GlcNAc. The acceptor specificity of recombinant full-length, membrane-bound fucosyltransferases, expressed in COS-7 cells, and soluble, protein-A chimeric forms of α1,3-fucosyltransferase (Fuc-T) III, Fuc-TIV, and Fuc-TV was analyzed toward a broad panel of oligosaccharide, glycolipid, and glycoprotein substrates. Our results on the full-length enzymes confirm and extend previous studies. However, chimeric Fuc-Ts showed increased activity toward glycoproteins, whereas chimeric Fuc-TIII and Fuc-TV had a decreased activity with glycosphingolipids, compared to the full-length enzymes. Unexpectedly, chimeric Fuc-TV exhibited a GDP-fucose hydrolyzing activity. In substrates with multiple acceptor sites, the preferred site of fucosylation was identified. Fuc-TIII and Fuc-TV catalyzed fucose transfer exclusively to OH-3 of glucose in lacto-N-neotetraose and lacto- N-tetraose, respectively, as was demonstrated by 1H NMR spectroscopy. Thin layer chromatography immunostaining revealed that FucT-IV preferred the distal GlcNAc residue in nLc6Cer, whereas Fuc-TV preferred the proximal GlcNAc residue. Incubation of Fuc-TIV or Fuc-TV with VI3NeuAcnLc6Cer resulted in products with the sialyl-LewisXepitope as well as the VIM-2 structure. To identify polar groups on acceptors that function in enzyme binding, deoxygenated substrate analogs were tested as acceptors. All three Fuc-Ts had an absolute requirement for a hydroxyl at C-6 of galactose in addition to the accepting hydroxyl at C-3 or C-4 of GlcNAc. Many antigenic carbohydrates on the surface of cells, including those on glycoproteins and glycolipids, contain fucose residues in α1,3 or α1,4 linkage to subterminal and/or internal GlcNAc residues. They are often transiently expressed at defined stages of development (1Solter D. Knowles B.B. Proc. Natl. Acad. Sci. U.S.A. 1978; 75: 5565-5569Crossref PubMed Scopus (1123) Google Scholar, 2Fenderson B.A. Holmes E.H. Fukushi Y. Hakomori S. Dev. Biol. 1985; 114: 12-21Crossref Scopus (78) Google Scholar, 3Feizi T. Childs R.A. Biochem. J. 1987; 245: 1-11Crossref PubMed Scopus (221) Google Scholar–4Gooi H.C. Feizi T. Kapadia A. Knowles B.B. Solter D. Evans M.J. Nature. 1981; 292: 156-158Crossref PubMed Scopus (489) Google Scholar) and are involved in the process of compaction during embryogenesis (4Gooi H.C. Feizi T. Kapadia A. Knowles B.B. Solter D. Evans M.J. Nature. 1981; 292: 156-158Crossref PubMed Scopus (489) Google Scholar). Because fucosylated glycoproteins and glycolipids also accumulate in a large variety of human cancers (5Hakomori S. Adv. Cancer Res. 1989; 52: 257-331Crossref PubMed Scopus (1076) Google Scholar, 6Hakomori S. Nudelman E. Levery S.B. Kannagi R. J. Biol. Chem. 1984; 259: 4672-4680Abstract Full Text PDF PubMed Google Scholar–7Feizi T. Nature. 1985; 314: 53-57Crossref PubMed Scopus (1013) Google Scholar), these structures are regarded as oncodevelopmental antigens. Furthermore, the sialyl-LewisX (sLeX) 1The abbreviations used are:sLeXsialyl-LewisX (NeuAc α 2⟶3Galβ1⟶4[Fuc α 1⟶3]GlcNAc)α1-AGPα1-acid glycoproteinCerceramideFuc-T α1,3-fucosyltransferaseGDP-Fuc:Galβ1⟶4GIcNAc⟶Rα(1⟶3)fucosy l transferaselacNAcN-acetyllactosamine (Galβ1⟶4GlcNAc)Lclacto-series glycolipidLeXLewisX (Gaβ1⟶4[Fucα1⟶3]GlcNAc)LNFlacto-N-fucopentaose (Galβ1⟶3[Fucα1⟶4]GlcNAcβ1⟶3Galβ1⟶4Glc)LNnFlacto-N-neofucopentaose (Galβ1⟶4[Fucαl⟶3]GlcNAβ1⟶3Galβ1⟶4Glc)LNTlacto-N-tetraose (Galβ1⟶3GlcNAcβ1⟶3Galβ1⟶4Glc)LNnTlacto-N-neotetraose (Galβ1⟶4GlcNAcβ1⟶3Galβ1⟶4Glc)NEMN-ethylmaleimidenLcneolacto-series glycolipidPCRpolymerase chain reactionTLCthin layer chromatographyBSAbovine serum albuminppmparts/millionMOPS4-morpholinepropanesulfonic acid. 1The abbreviations used are:sLeXsialyl-LewisX (NeuAc α 2⟶3Galβ1⟶4[Fuc α 1⟶3]GlcNAc)α1-AGPα1-acid glycoproteinCerceramideFuc-T α1,3-fucosyltransferaseGDP-Fuc:Galβ1⟶4GIcNAc⟶Rα(1⟶3)fucosy l transferaselacNAcN-acetyllactosamine (Galβ1⟶4GlcNAc)Lclacto-series glycolipidLeXLewisX (Gaβ1⟶4[Fucα1⟶3]GlcNAc)LNFlacto-N-fucopentaose (Galβ1⟶3[Fucα1⟶4]GlcNAcβ1⟶3Galβ1⟶4Glc)LNnFlacto-N-neofucopentaose (Galβ1⟶4[Fucαl⟶3]GlcNAβ1⟶3Galβ1⟶4Glc)LNTlacto-N-tetraose (Galβ1⟶3GlcNAcβ1⟶3Galβ1⟶4Glc)LNnTlacto-N-neotetraose (Galβ1⟶4GlcNAcβ1⟶3Galβ1⟶4Glc)NEMN-ethylmaleimidenLcneolacto-series glycolipidPCRpolymerase chain reactionTLCthin layer chromatographyBSAbovine serum albuminppmparts/millionMOPS4-morpholinepropanesulfonic acid. determinant (NeuAcα2⟶3Galβ1⟶4[Fucαl⟶3]-GlcNAc) has been identified as an important carbohydrate ligand for a family of adhesion molecules, the selectins (8Philips M.L. 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Commun. 1991; 181: 1218-1222Crossref PubMed Scopus (69) Google Scholar). sialyl-LewisX (NeuAc α 2⟶3Galβ1⟶4[Fuc α 1⟶3]GlcNAc) α1-acid glycoprotein ceramide GDP-Fuc:Galβ1⟶4GIcNAc⟶Rα(1⟶3)fucosy l transferase N-acetyllactosamine (Galβ1⟶4GlcNAc) lacto-series glycolipid LewisX (Gaβ1⟶4[Fucα1⟶3]GlcNAc) lacto-N-fucopentaose (Galβ1⟶3[Fucα1⟶4]GlcNAcβ1⟶3Galβ1⟶4Glc) lacto-N-neofucopentaose (Galβ1⟶4[Fucαl⟶3]GlcNAβ1⟶3Galβ1⟶4Glc) lacto-N-tetraose (Galβ1⟶3GlcNAcβ1⟶3Galβ1⟶4Glc) lacto-N-neotetraose (Galβ1⟶4GlcNAcβ1⟶3Galβ1⟶4Glc) N-ethylmaleimide neolacto-series glycolipid polymerase chain reaction thin layer chromatography bovine serum albumin parts/million 4-morpholinepropanesulfonic acid. sialyl-LewisX (NeuAc α 2⟶3Galβ1⟶4[Fuc α 1⟶3]GlcNAc) α1-acid glycoprotein ceramide GDP-Fuc:Galβ1⟶4GIcNAc⟶Rα(1⟶3)fucosy l transferase N-acetyllactosamine (Galβ1⟶4GlcNAc) lacto-series glycolipid LewisX (Gaβ1⟶4[Fucα1⟶3]GlcNAc) lacto-N-fucopentaose (Galβ1⟶3[Fucα1⟶4]GlcNAcβ1⟶3Galβ1⟶4Glc) lacto-N-neofucopentaose (Galβ1⟶4[Fucαl⟶3]GlcNAβ1⟶3Galβ1⟶4Glc) lacto-N-tetraose (Galβ1⟶3GlcNAcβ1⟶3Galβ1⟶4Glc) lacto-N-neotetraose (Galβ1⟶4GlcNAcβ1⟶3Galβ1⟶4Glc) N-ethylmaleimide neolacto-series glycolipid polymerase chain reaction thin layer chromatography bovine serum albumin parts/million 4-morpholinepropanesulfonic acid. The biosynthesis of fucose-containing glycoconjugates requires the action of several glycosyltransferases, of which fucosylation is the last and critical step (18Holmes E.H. Ostrander G.K. Hakomori S. J. Biol. Chem. 1986; 261: 3737-3743Abstract Full Text PDF PubMed Google Scholar, 19Hanisch F.G. Mitsakos A. Schroten H. Uhlenbruck G. Carbohydr. Res. 1988; 178: 23-28Crossref PubMed Scopus (18) Google Scholar). The individual members of the fucosyltransferase (Fuc-T) family are discriminated by differences in substrate specificities, cation requirement, sensitivity to inhibitors, and tissue distribution (20Mollicone R. Gibaud A. Francois A. Ratcliffe M. Oriol R. Eur. J. Biochem. 1990; 191: 169-176Crossref PubMed Scopus (126) Google Scholar, 21Macher B.A. Holmes E.H. Swiedler S.J. Stults C.L.M. Srnka C.A. Glycobiology. 1991; 1: 577-584Crossref PubMed Scopus (83) Google Scholar–22de Vries Th. van den Eijnden D.H. Histochem. J. 1992; 24: 761-770Crossref PubMed Scopus (49) Google Scholar). A precise definition of which enzyme(s) is expressed in individual tissues or cell types is complicated by the possibility of multiple fucosyltransferases present therein. Several cDNAs coding for Fuc-Ts have been cloned (23Kukowska-Latallo J.F. Larsen R.D. Nair R.P. Lowe J.B. Genes & Dev. 1990; 4: 1288-1303Crossref PubMed Scopus (467) Google Scholar, 24Lowe J.B. Kukowska-Latallo J.F. Nair R.P. Larsen R.D. Marks R.M. Macher B.A. Kelly R.J. Ernst L.K. J. Biol. Chem. 1991; 266: 17467-17477Abstract Full Text PDF PubMed Google Scholar, 25Goelz S.E. Hession C. Goff D. Giffiths B. Tizard R. Newman B. Chi-Rosso G. Lobb R. Cell. 1990; 63: 1349-1356Abstract Full Text PDF PubMed Scopus (285) Google Scholar, 26Kumar R. Potvin B. Muller W.A. Stanley P. J. Biol. Chem. 1991; 266: 21777-21783Abstract Full Text PDF PubMed Google Scholar, 27Weston B.W. Nair R.P. Larsen R.D. Lowe J.B. J. Biol. Chem. 1992; 267: 4152-4160Abstract Full Text PDF PubMed Google Scholar, 28Weston B.W. Smith P.L. Kelly R.J. 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Chem. 1994; 269: 12662-12671Abstract Full Text PDF PubMed Google Scholar), and Fuc-TVII, a leukocyte-expressed fucosyltransferase (30Sasaki K. Kurata K. Funayama K. Nagata M. Watanabe E. Ohta S. Hanai N. Nishi T. J. Biol. Chem. 1994; 269: 14730-14737Abstract Full Text PDF PubMed Google Scholar, 31Natsuka S. Gersten K.M. Zenita K. Kannagi R. Lowe J.B. J. Biol. Chem. 1994; 269: 16789-16794Abstract Full Text PDF PubMed Google Scholar). Recombinant forms of these enzymes allow study of a single enzyme species. The limited studies of acceptor specificity performed to date allow only a partial assignment of a correspondence between cloned enzymes and ones in tissues and cell lines. Our goal was to characterize the enzymatic properties of recombinant forms of Fuc-TIII, Fuc-TIV, and Fuc-TV. Substrate preferences were determined with a matrix of acceptor substrates including oligosaccharides, glycolipids, and glycoproteins. To determine which functional groups on an acceptor are crucial for enzyme recognition, each species of recombinant Fuc-T was analyzed for its ability to transfer fucose to deoxy analogs of acceptors. Recombinant Fuc-Ts share significant amino acid sequence homology (27Weston B.W. Nair R.P. Larsen R.D. Lowe J.B. J. Biol. Chem. 1992; 267: 4152-4160Abstract Full Text PDF PubMed Google Scholar), despite differences in acceptor specificity. As approximately 90% of the amino acid differences between Fuc-TIII and Fuc-TV are confined to the N-terminal part of the proteins (28Weston B.W. Smith P.L. Kelly R.J. Lowe J.B. J. Biol. Chem. 1992; 267: 24575-24584Abstract Full Text PDF PubMed Google Scholar), it has been suggested that this domain directs the acceptor specificity. We evaluated this possibility by examining the acceptor specificity of Fuc-Ts truncated in this region by selecting areas that were approximately equally spaced from the transmembrane domain and each other. Constructs encoding amino acids 44–361 and 52–361 of Fuc-TIII were amplified by PCR and subcloned into a vector, which contained a protein A coding sequence preceding the point of insertion, to produce protein A-Fuc-T fusion proteins. Similar constructs were prepared for Fuc-TV (containing amino acids 44–374 and 62–374) and for Fuc-TIV (containing amino acids 58–405). These shortened protein A-Fuc-T chimeric proteins were compared to full-length recombinant enzymes. An aim of this study is to provide insight into structure-function relationships of the fucosyltransferases encoded by the human genome. Significant effort is underway to block selectin-ligand interactions and to identify inhibitors of α1,3-fucosyltransferases to limit the synthesis of selectin ligands, and thus produce anti-inflammatory and possibly anti-metastatic drugs. Knowledge about the active sites of the Fuc-Ts may allow design of effective inhibitors. Moreover, soluble secreted forms of Fuc-Ts will probably be the likely candidates for use in large scale synthesis of potential selectin inhibitors. Therefore, a thorough knowledge of these forms of Fuc-Ts is required. GDP-Fucose and compounds 2, 4, 6, 9, 11, and 12 were kind gifts of Dr. Ole Hindsgaul (University of Alberta, Edmonton, Alberta). Compounds 21–28 were kindly donated by Dr. R. U. Lemieux (University of Alberta, Edmonton, Alberta). Compounds 29–31 were the generous gifts of Dr. O. Srivastava (Alberta Research Council, Edmonton, Alberta). N-Acetyllactosamine (lacNac), 1; lacto-N-biose I, 8; lactose, 13; 2′-fucosyllactose, 14 and 3′-sialyllactose, 15 were purchased from Sigma; lacto-N-neotetraose (LNnT), 3; 3′-sialyl-lacNAc, 5; 6′-sialyl-lacNAc, 7 and lacto-N-tetraose (LNT), 10 were purchased from Oxford GlycoSystems. Neutral glycosphingolipids (structures 16–18) were prepared from sheep thymus (33He P. Hu J. Macher B.A. Arch. Biochem. Biophys. 1993; 305: 350-361Crossref PubMed Scopus (12) Google Scholar), as described previously. Gangliosides (19 and 20) were isolated from chronic myelogenous leukemia cells as described (34Westrick M.A. Lee W.M.F. Macher B.A. Cancer Res. 1983; 43: 5890-5894PubMed Google Scholar). α1-Acid glycoprotein (α1-AGP) was isolated from human plasma (35Hao Y.-L. Wickerhauser M. Biochim. Biophys. Acta. 1973; 322: 99-108Crossref PubMed Scopus (60) Google Scholar). Calf fetuin was obtained from Life Technologies, Inc. Asialo-α1-AGP and asialo-fetuin were prepared by mild acid hydrolysis (0.1 M trifluoroacetic acid, 1 h, 80 °C) of the corresponding native glycoprotein. GDP-[14C]Fuc (250 Ci/mol) was purchased from New England Nuclear. Antibody 1B2 (36Young W.W. Portoukalial J. Hakomori S. J. Biol. Chem. 1981; 256: 10967-10972Abstract Full Text PDF PubMed Google Scholar) was a gift of Dr. R. Mandrell (Veterans Administration Medical Center, San Francisco, CA). My-1 (37Huang L.C. Civin C.I. Magnani J.L. Shaper J.H. Ginsberg V. Blood. 1983; 61: 1020-1023Crossref PubMed Google Scholar) was a gift of Dr. C. Civin (Johns Hopkins School of Medicine, Baltimore, MD). CSLEX-1 (38Fukushima K. Hirota M. Terasaki P.I. Wakisaka A. Togashi H. Chia D. Suyama H. Fukushi Y. Nudelman E. Hakomori S. Cancer Res. 1984; 44: 5279-5285PubMed Google Scholar) was obtained from Becton-Dickinson (Mountain View, CA) and VIM-2 (39Macher B.A. Buehler J. Scudder P. Knapp W. Feizi T. J. Biol. Chem. 1988; 263: 10186-10191Abstract Full Text PDF PubMed Google Scholar) was a gift from Dr. W. Knapp (University of Vienna). Protein assay reagent was obtained from Bio-Rad. Hydrofluor liquid scintillation mixture was obtained from National Diagnostics. All other chemicals were obtained from commercial sources and were of the highest purity available. Full-length copies of cDNA encoding Fuc-TIII, Fuc-TIV, and Fuc-TV (23Kukowska-Latallo J.F. Larsen R.D. Nair R.P. Lowe J.B. Genes & Dev. 1990; 4: 1288-1303Crossref PubMed Scopus (467) Google Scholar, 24Lowe J.B. Kukowska-Latallo J.F. Nair R.P. Larsen R.D. Marks R.M. Macher B.A. Kelly R.J. Ernst L.K. J. Biol. Chem. 1991; 266: 17467-17477Abstract Full Text PDF PubMed Google Scholar, 27Weston B.W. Nair R.P. Larsen R.D. Lowe J.B. J. Biol. Chem. 1992; 267: 4152-4160Abstract Full Text PDF PubMed Google Scholar) were produced by PCR using nested primers directed to flanking sequences and ligated into the vector pCDM8 prior to amplification in bacteria. Truncated forms of the DNA encoding the fucosyltransferases (shortened at the 5′ ends) were similarly prepared by PCR from verified full-length constructs and ligated into a plasmid which contains a protein A sequence preceding the point of insertion (40Wei Z. Swiedler S.J. Ishihara M. Orellana A. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 90: 3885-3888Crossref Scopus (66) Google Scholar). For truncated Fuc-TIII, nucleotides 130 and 157 of the translated Fuc-TIII sequence were starting points for, respectively, long (Fuc-TIIIl) and medium (Fuc-TIIIm) forms. For truncated Fuc-TIV, nucleotide 172 was a starting point for the long (Fuc-TIVl) form. For truncated Fuc-TV, nucleotides 130 and 184 were starting points for long (Fuc-TVl) and medium (Fuc-TVm) forms. A DNA sequence encoding protein A was connected by a polylinker to the 5′ end of the truncated Fuc-T in each plasmid, resulting in production of a chimeric Fuc-T during translation. Following selective amplification in bacteria, plasmids with desired DNA constructs encoding Fuc-Ts and chimeric Fuc-Ts were purified by ultracentrifugation on cesium chloride gradients. Plasmid DNA was dissolved in sterile water at 1 μg/μl before use in transfection. The DNA inserts were verified by sequence analysis by dideoxy chain termination (41Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52251) Google Scholar), using T7 DNA polymerase (Pharmacia Biotech). DNA was transfected into COS cells by a DEAE-dextran protocol, using from 2 to 10 μg of DNA/100-mm tissue culture dish seeded with 0.4 × 106 cells (42Davis L.G. Dibner M.D. Battey J.F. Basic Methods in Molecular Biology. Elsevier Science Publishing Co., New York1986: 290-292Crossref Google Scholar). COS cell clones expressing the full-length constructs of Fuc-Ts were identified by antibody-linked immunofluorescent display of appropriate fucosylated antigens on the COS cell surface 3 days after transfection. COS cells mock-transfected with irrelevant DNA displayed only background levels of immunofluorescence. Chimeric Fuc-Ts were isolated from the media of similarly transfected COS cells on days 2 through 6 post-transfection, using rabbit IgG-agarose to purify the protein A-Fuc-T chimeras. All enzyme preparations were done on ice. To elute chimeric Fuc-Ts from IgG-agarose beads, the beads (10 μl) were washed with 1 ml of 10mM Tris-HCl, pH 8, containing 150 mM NaCl, and pelleted. The supernatant was removed and 143 μl of 0.1 M sodium citrate, pH 4.4, was added. The tube was vortexed for 5 s, incubated for 1 min, and centrifuged. The supernatant was added to 57 μl of 1 M Tris-HCl, pH 8.2, containing 0.1% BSA. Released enzyme was diluted 4-fold with buffer A (50 mM cacodylate, pH 7.2, containing 50% glycerol, 100 mM NaCl, 5 mM MgCl2, 0.1% BSA, and 0.05% sodium azide) and stored at 4 °C until use. The resulting enzyme preparation was stable for at least 6 months. Activity of the Fuc-T-protein A chimeras is expressed as picomoles˙minute−1˙milliliter−1 (the addition of a carrier protein, BSA, to ensure recovery of Fuc-T protein precluded protein determination of Fuc-T). Thawed pellets of transfected COS-7 cells were resuspended in 50mM cacodylate, pH 6.7, to a protein concentration of 8 – 10 μg/μl. Extracts were prepared as described (43Robinson N.E. de Vries Th. Davis R.E. Stults C.L.M. Watson S.R. van den Eijnden D.H. Macher B.A. Glycobiology. 1994; 4: 317-326Crossref PubMed Scopus (18) Google Scholar). The standard reaction mixture contained in 50 μl: 5 nmol of GDP-[14C]Fuc (4–5 Ci/mol), 2.5 μ mol of MOPS/NaOH, pH 7.5, 1.0 μmol of MnCl2, 5 μmol of NaCl, 0.2 μmol of ATP, 50 nmol of acceptor (unless stated otherwise), and an amount of enzyme preparation corresponding to initial velocity and giving similar fucose transfer for each preparation. Reaction mixtures were incubated for 1 h (chimeras) or 4 h (cell extracts). The reaction was stopped by dilution with 200 μl of cold water. The mixture and two additional rinses (200 μl) were applied to a column of 1 ml of Dowex 1-X2 (200–400 mesh, Cl− form). The column was washed with 2 ml of water and the radioactivity in the effluent measured. The column was eluted with 2 ml of 0.5 m acetic acid when sialylated oligosaccharides were used as acceptors. Values were corrected for incorporation into endogenous acceptors. Three replicates were used per data point. A unit of activity was defined as the amount of enzyme catalyzing the transfer of 1μmol of fucose˙min−1 to an acceptor substrate under standard assay conditions. Acceptor substrates (18.7 nmol) were incubated in the standard reaction mixture for 2 h at 37 °C. Subsequently, the reaction was stopped by adding 200 μl of cold water, and the mixture was applied to a Sep-Pak C18 cartridge (44Palcic M.M. Heerze L.D. Pierce M. Hindsgaul O. Glycoconjugate J. 1988; 5: 49-63Crossref Scopus (277) Google Scholar). Two additional rinses (200 μl) were also applied to the Sep-Pak. The cartridge was washed with H2O (3 × 5 ml) and the product eluted with methanol (5 ml). The eluate was dried and the incorporation of [14C]fucose determined by scintillation counting. 0.25 mg of α1-AGP or 0.5 mg of fetuin (native or asialo, 110 nmol of acceptor sites calculated from galactose content of the protein) were incubated in the standard reaction mixture for 1 h at 37 °C. The reaction was stopped by adding 200 μl of cold 1% BSA in water. Incorporation of fucose into the glycoprotein was determined by acid precipitation as described (45Joziasse D.H. Bergh M.L.E. ter Hart H.G.J. Koppen P. Hooghwinkel G.J.M. van den Eijnden D.H. J. Biol. Chem. 1985; 260: 4941-4951Abstract Full Text PDF PubMed Google Scholar). Twenty μg of glycolipid in 20 μl of 85% ethanol and 5 μl of 5% sodium taurodeoxycholate in CHCl3/CH3 OH (1:1) were dried together at room temperature in a microtiter well. The standard reaction mixture was added together with an amount of enzyme preparation. Before incubation the microtiter plate was sonicated for 1 min to allow the formation of glycolipid/sodium taurodeoxycholate micelles. Incubation was performed for 8.5 h (chimeras) or 24 h (cell extracts). The reaction products were recovered from the wells as follows: 150 μl of 85% ethanol was added to the well, mixed thoroughly with the incubation mixture, and the total mixture was applied to a Sep-Pakcartridge. Wells were rinsed twice with 100 μl of 85% ethanol, rinses were diluted with 5 ml of water, and applied to the Sep-Pak cartridge. The cartridge was washed, and the incorporation of [14C]fucose into the glycolipid was determined. The apparent Km for GDP-fucose was determined at 500 μM of compound 11 for Fuc-TIII and 500 μM of compound 4 for Fuc-TIV and Fuc-TV under the above assay conditions. Apparent kinetic parameters for the acceptor substrates were determined using a close to saturating concentration of GDP-fucose (50 μM). The kinetic parameters were determined using SigmaPlot (Jandel scientific) and fit to the Michaelis-Menten curve. Sensitivity toward NEM (final concentration in the reaction mixture was 10 mM) was determined by preincubation of the enzyme preparation with NEM for 1 h at 0 °C. One-ml suspensions of "enzyme on beads" (Fuc-TVm or Fuc-TIIIm) was treated as above to elute the enzyme from the IgG-beads. The eluted enzyme was added directly to a tube containing: 500 nmol of 10 (for Fuc-TVm), 1500 nmol of GDP-[14C]Fuc (0.2 Ci/mol), 20 μmol of MnCl2, and 4 μmol of ATP or 200 nmol of 3 (for Fuc-TIIIm), 600 nmol of GDP-[14C]Fuc (0.2 Ci/mol), 20μmol of MnCl2, and 4μmol of ATP. Incubation was conducted for 48 h at 37 °C. Each reaction mixture was applied to a 1-ml Dowex 1-X8 (100–200 mesh) column, which was washed with 4 ml of water, and the flow-through was lyophilized. The residual material was dissolved in 500 μl of 50 mM ammonium acetate, pH 5.2, applied to a column (1.6 × 200 cm) of Bio-Gel P-4 (200–400 mesh) equilibrated with, and eluted at a flow of 14 ml/h with 50 mM ammonium acetate, pH 5.2, at 45 °C. Fractions (4 ml) were collected, monitored for radioactivity, pooled (fraction 67–71), lyophilized, and desalted on a column (1 × 40 cm) of Bio-Gel P-2 (200–400 mesh) eluted with water. Yields were 220 nmol of fucosylated 10 (44% relative to acceptor substrate) and 84 nmol of fucosylated 3 (42% relative to acceptor substrate). For structural characterization of fucosylated glycolipid products (i.e. Fuc-TIV, with nLc6 Cer (18) or VI3 NeuAcnLc6 Cer (20) and Fuc-TVm with nLc6 Cer or VI3 NeuAcnLc6 Cer), incubations were performed for 24 h at 37 °C. Two wells were pooled for the reactions of Fuc-TIV1, with nLc6Cer and NeuAcnLc6Cer and four wells for the reactions of Fuc-TVm. The products and unreacted glycolipid substrate were separated from the other components of the reaction mixture by Sep-Pak C18 chromatography. The methanol phase, containing glycosphingolipids, was dried under a stream of nitrogen, dissolved in 500 μl of chloroform/methanol (1:1), and dried again. The sample was redissolved in 50 μl of chloroform/methanol (1:1) and spotted on an aluminum-backed Silica Gel 60 TLC plate and chromatographed in chloroform/methanol/water (60:35:8). Immunostaining was performed as described (46Buehler J. Macher B.A. Anal. Biochem. 1986; 158: 283-287Crossref PubMed Scopus (40) Google Scholar), with 1B2, My-1, CSLEX-1, or VIM-2 as the primary antibody. Prior to 1H NMR spectroscopic analysis, the acceptor substrates (compounds 3 and 10) and the fucosylated products (100–300 nmol) were exchanged in D2O three times, with intermediate lyophilization. Finally, each sample was redissolved in 400 μl of D2O (99.96 atom% D, Aldrich). 1H NMR spectroscopy was performed on a Bruker MSL 400 spectrometer operating at 400 MHz at a probe temperature of 300 K. Resolution enhancement was achieved by Lorentzian to Gaussian transformation. Chemical shifts are expressed in ppm downfield from internal sodium 4,4-dimethyl-4-silapentane-1-sulfonate but were actually measured by reference to internal acetone (δ 2.225 in D2O). Various forms of Fuc-Ts (full-length and N-terminal truncated chimeras) were tested with different acceptors. In most cases, the acceptor specificity of both full-length and chimeric Fuc-Ts was determined. For truncated forms of enzymes, the following constructs were assayed: Fuc-TIIIl, containing amino acids 44–361; Fuc-TIIIm, 52–361; Fuc-TIVl, 58–405; Fuc-TVl, 44–374; and Fuc-TVm, 62–374, all as chimeras with protein A. However, some forms of a given enzyme lacked activity with a particular class of acceptors (see below). Some significant differences were found in the acceptor specificity of full-length versus chimeric forms of Fuc-Ts, and these novel observations are described in a subsequent section (see below). The acceptor specific
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